Our current understanding of brain function is limited by available experimental techniques. In particular, methods for measuring in vivo neurochemical dynamics are lacking. This is critical considering that it is these dynamics that are the primary mechanism of the neuronal communication that gives rise to brain function. Microdialysis is currently the dominant technique for monitoring in vivo chemical dynamics. Unfortunately, microdialysis suffers from some significant drawbacks. Temporal resolution is often on the order of 10-20 minutes. Sampling regions are large (1-5 mm) making it difficult to measure activity in specific brain regions. Damage during implantation is significant and rigorous calibration is not possible. Introduction of online microdialysis - capillary electrophoresis (MD-CE) assays have improved temporal resolution to 10- 30 s but many issues remain. We propose to systematically overhaul existing online MD-CE instrumentation with the goal of achieving significant gains in temporal and spatial resolution. Micro-free flow electrophoresis (u-FFE) will be introduced as a separation technique capable of continuously analyzing sampled neurotransmitters. The sample stream will be compartmentalized using an immiscible fluorous solvent to prevent loss of temporal information as neurotransmitters travel from the probe, through the online labeling reaction and to the separation interface. An electrically actuated, inline micropump will be developed to directly sample fluid from the rat brain at very low flow rates (<50 nL/min). The spatial resolution of this direct sampling technique will only be limited by the outlet size of the sampling capillary (40 um). Combined we expect an online direct sampling - uFFE instrument will be capable of monitoring the in vivo dynamics of a number of important neurotransmitters, including glutamate, gaba, glycine, D-serine and taurine, on a -100 ms time scale in <100 um brain regions. Measurements on this time and size scale for a range of neurotransmitters would impact all areas of neuroscience research including stroke, epilepsy, memory, pain, addiction, etc. As an added benefit the instrumentation described in this proposal is much simpler than existing high temporal resolution microdialysis assays, suggesting that it may be more easily adopted by non-specialists in traditional neuroscience laboratories.